There are a wide variety of assays available in the art, including, but not limited to, those that assay for the presence or absence or amount of a polynucleotide, protein, organism, or other molecular species and the like.
Recent efforts have attempted to employ waveguide technology in performing such assays. The presence and/or absence and/or quantity of a material to be analyzed (the “analyte”) is determined by use of fluorescent material that functions as a reporter with the fluorescent material being excited during the assay and the light emitted from the fluorescent material being directed to a detector by the use of a waveguide.
One such example is an optrode. (see refs. 1–3). The optrode is an optical fiber having probe molecules, typically antibodies, immobilized at its distal ends. Excitation light is delivered through the fiber to the probe-target-reporter complexes that form at the end of the fiber. The resultant fluorescent emission is wave-guided back up the fiber to an optical system that delivers the emitted light to a photomultiplier tube or other detector. The key role of the fiber in this sensor is that both excitation and emission light is guided through the fiber very efficiently due to the phenomenon of total internal reflection (TIR). Optical fibers are specially designed so that the index of refraction of the fiber (nf) is greater than the index of refraction of the material cladding the fiber (nc). When light, introduced into the fiber from any source, strikes the fiber-cladding interface, it will be reflected with essentially 100% efficiency, due to the phenomenon of TIR, as long as the angle of incidence is less than the critical angle (θc), which is defined asθc=sin−1(nc/nf).
Sensitivity of such sensors is greatly enhanced by the efficiency with which light is guided via TIR from the probe-derivatized surface of the sensor, through the fiber, to the detector.
Recently this basic approach has been expanded upon to develop manifolds in which several probes are applied as small spots on the distal surface of a single fiber optic. Optical systems were designed to assess interactions between probes and targets individually. A similar, but more powerful system has been devised in which micron-scale optical fibers are configured as a manifold in which indentations at the end of the manifold receive small beads of different colors, each of which is derivatized with a different oligonucleotide probe, and to which the corresponding target and reporter are bound (4). This system provides a reusable fiber-optical manifold that can be used to assess hybridization of any set of targets to their respective probes.
Optrodes have found applications in a number of areas, but have significant limitations in that the area is limited to which probe molecules can be coupled. This limits the sensitivity of the sensor. Another limitation is that fluorescence emitted from any molecule located in the vicinity of the sensing terminus of the fiber will be picked up by the fiber, which contributes significant levels of background noise.
Another sensor is an evanescent wave-based sensor (1,5,6). When light strikes the interface between the optical fiber and the surrounding medium, which is of lower index of refraction, it undergoes total internal reflection. However, an electromagnetic component of the light passes through the interface, and is propagated through the surrounding medium in a direction parallel to the fiber. This is called the evanescent wave. It penetrates only a short distance (a fraction of the wavelength of the light used) into the medium surrounding the fiber, decaying exponentially as a function of the wavelength of the light. However, this wave can effectively excite fluorescent compounds located close to the fiber surface.
Although other designs have been devised (7), the most common design for evanescent wave-based sensors is to immobilize probe molecules on the walls (not the distal end) of the fiber. Probe-target-reporter complexes formed on the surface of the fiber are detected when the evanescent wave excites the reporter fluor molecules, which emit fluorescence, and which when it strikes the fiber wall at appropriate angles will enter the fiber and be wave-guided up the fiber to the detector.
This design has the advantage over the optrode that fluor molecules in the bulk solution surrounding the fiber are not excited because the evanescent wave does not propagate significantly into the bulk solution phase. Only free fluors that happen to be located within about 0.5 wavelength of the fiber wall will be excited. This reduces background fluorescence that is picked up by the bulk solution surrounding the fiber.
The disadvantage of this approach is that the power of the evanescent wave is at most 2% of the power of the excitation light within the fiber. Thus, effective excitation of the probe-target-reporter complexes can be challenging. Similarly, a large proportion of the fluorescence emitted by probe-target-reporter complexes fails to be coupled into the fiber, because of the unfavorable geometry of the system. Both of these features limit the sensitivity and utility of evanescent wave sensors.
Another sensor involves the use of non-evanescent-wave-based fiber optic systems (8). The limitation with evanescent wave-based sensors is the inefficiency with which excitation light can be transmitted out of the fiber to the probe-target-reporter layer, and the inefficiency with which fluorescence is coupled back into the fiber to be waveguided to the detector. Other mechanisms have been used in attempts to increase these efficiencies. For instance, attempts have been made to match the index of refraction of the probe layer with that of the fiber. The intention in this design is to include the probe layer within the waveguide so that a larger portion of the exciting light reaches probe-target-reporter complexes, and a larger portion of emitted fluorescence is coupled into the waveguide. Empirical evidence indicates that fibers constructed based on this principle function with somewhat improved efficiency compared to evanescent wave-based sensors.
A further example is a surface plasmon resonance-based sensor (1,9,10). Sensors based on the surface plasmon resonance phenomenon have the advantage that they directly detect changes in index of refraction at the surface of the optical system due to binding of target to probe. Thus, this method does not require the use of fluorescent or other reporter molecules.
This optical system consists in simplest form of a prism, one surface of which is coated with a thin metal film (usually gold or silver). Probe molecules are attached to the other surface of the metal film, which is also in contact with a solution that may contain the analyte or target of interest.
The evanescent wave generated by light impinging on the prism-metal interface at a certain incident angle, termed the resonant angle or SPR angle, will couple with and excite the free-electron plasma of the metal film. That is, electromagnetic coupling occurs between the free electrons of the metal film and the evanescent wave of the light that undergoes total internal reflection at the SPR angle within the prism. This generates a resonant wave that propagates along the surface of the metal. Dissipative processes within the metal film absorb some of the energy of this resonant wave. As a result, the light incident upon the prism-metal interface at the SPR angle is reflected with attenuated intensity. In other words, at the SPR angle, light energy is transformed into dissipative surface plasmons within the metal. This is observed as attenuation of the total internal reflection in the prism.
The SPR angle depends not only on the properties of the metal film but also on the dielectric constant (and thus the index of refraction) of the medium immediately adjacent (within a fraction of one wavelength) to the other surface of the metal film. This is because of interaction between the evanescent field of the SPR wave and the medium immediately adjacent to the metal film. Materials that bind to the surface of the metal film alter the dielectric constant (and index of refraction) in that zone, altering the SPR angle. This is observed as a change in the angle at which total internal reflection is attenuated.
This change in the SPR angle is a sensitive indicator of binding to the metal surface, and can act as a sensor for specific molecular species (target molecules) present in a solution that bathes the metal surface. To accomplish this, probe molecules are bound to the metal film. When target molecules are present in the solution bathing the metal film, they form complexes with the immobilized probe. This changes the index of refraction immediately adjacent to the metal surface, which, in turn, alters the SPR angle. The change in SPR angle can be directly measured. With proper calibration, such a system can be used to directly and quantitatively measure the formation of probe-target complexes.